The synthesized CEA-specific nanoMIPs and their corresponding non-imprinted control particles (nanoNIPs) were characterized by DLS and zeta potential measurements as demonstrated in Fig. 3a,b, respectively. The hydrodynamic diameters of the nanoMIPs and nanoNIPs were 168.2 ± 25.5 nm and 159.5 ± 30.8 nm, respectively. The slightly larger size of the nanoMIPs was attributed to the molecular imprinting process. The polydispersity index (PDI) values of the nanoMIPs and nanoNIPs were 0.686 and 0.635, respectively, indicating moderate particle dispersity. The surface charge and colloidal stability of the synthesized nanoMIPs and nanoNIPs were evaluated by zeta potential measurement; these were found to be -56.1 ± 2.0 mV and - 62.8 ± 2.5 mV for the nanoMIPs and nanoNIPs, respectively. Negative zeta potential measurements represent the negative charge of the carboxyl groups present in the acrylic acid monomer. The slightly more negative zeta potential observed in the nanoNIPs may be due to structural differences resulting from the absence of the molecular imprinting process. Finally, high negative zeta potential values are indicative of strong electrostatic repulsion between particles, contributing to their colloidal stability in solution.
The surface morphology of the modified electrodes was analyzed using SEM. SEM images of bare SPCE, MWCNTs/SPCE, OH/MWCNTs/SPCE, APTES/OH/MWCNTs/SPCE, and nanoMIPs/APTES/OH/MWCNTs/SPCE are presented in Fig. 3c,g, respectively. The bare SPCE exhibited a rough surface composed of graphite flakes and the binding agent from the carbon ink. Following MWCNT deposition, the surface of the MWCNTs/SPCE displayed a spaghetti-like morphology characteristic of MWCNTs, indicating that they had been successfully physically adsorbed onto the SPCE surface. No obvious morphological differences were observed in the OH/MWCNTs/SPCE and APTES/OH/MWCNTs/SPCE compared to the MWCNTs/SPCE. However, there was a notable increase in surface roughness observed in the nanoMIPs/APTES/OH/MWCNTs/SPCE, which appeared to coat the spaghetti-like structure of the MWCNTs. These results confirm the successful stepwise modification of the SPCE surface with MWCNTs and nanoMIPs.
To characterize the electrochemical behavior of the modified electrodes, square wave voltammetry (SWV) was performed using a 2.5 mM [Fe(CN)] solution as the redox probe. The potential was scanned from - 0.4 to 0.7 V using an amplitude of 0.05 V, a step potential of 0.005 V, and a frequency of 5 Hz. All stepwise modifications, including bare SPCE, MWCNTs/SPCE, OH/MWCNTs/SPCE, APTES/OH/MWCNTs/SPCE, and nanoMIPs/APTES/OH/MWCNTs/SPCE, were electrochemically characterized; the results of these analyses are presented in Fig. 4a. All electrodes exhibited well-defined oxidation peaks of the redox probe; however, the current responses varied depending on their surface modifications. For example, the MWCNTs/SPCE exhibited a higher current response compared to the bare SPCE due to the high surface area and excellent conductivity of MWCNTs, which enhances electron transfer at the electrode surface. In contrast, the current response decreased after electrochemical oxidation in NaOH due to the negatively charged hydroxyl groups introduced during the activation step, which repelled the redox probe and hindered electron transfer on the OH/MWCNTs/SPCE. The current response increased following the successful preparation of APTES/OH/MWCNTs/SPCE and the enhancement of electron transfer efficiency. This phenomenon can be explained by the favorable electrostatic interaction between the positively charged protonated amino groups of APTES and the negatively charged anionic probe [Fe(CN)]. The current response decreased once again following the subsequent immobilization of nanoMIPs due to their polymeric structure, which impedes electron transfer at the electrode surface, resulting in lower current responses for the nanoMIPs/APTES/OH/MWCNTs/SPCE. The SWV results confirmed the successful assembly of MWCNTs, hydroxyl groups, APTES, and nanoMIPs on the electrode surface.
Following the successful modification of nanoMIPs on the electrode surface, their CEA detection capabilities were evaluated using SWV with a 2.5 mM [Fe(CN)] redox probe. The prepared nanoMIPs/APTES/OH/MWCNTs/SPCE and nanoNIPs/APTES/OH/MWCNTs/SPCE were tested with a CEA concentration of 5 ng/mL and compared to the background signal (i.e., 0 ng/mL CEA). The electrochemical results are presented in Fig. 4b and d. There was a clear decrease in current response observed in the nanoMIPs/APTES/OH/MWCNTs/SPCE upon exposure to 5 ng/mL CEA, while only a slight decrease was observed in the nanoNIPs/APTES/OH/MWCNTs/SPCE. The current change observed in the nanoMIPs-modified electrode was 14.22 µA -- this was approximately 4.8 times greater than the values obtained from the nanoNIPs-modified electrode (2.95 µA). This higher current change highlights the superior sensitivity of nanoMIPs in capturing the target CEA molecules. Furthermore, these results confirm the specific binding capabilities and sensing performance of the nanoMIPs-based electrochemical sensor for CEA detection developed in this study.
The surface morphology of the synthesized UiO-66-NH was characterized using SEM (Fig. 5a). The SEM images revealed that the surface of the UiO-66-NH exhibited an octahedral-like structure with uniform size and a smooth surface. Figure 5b presents the X-ray Diffraction (XRD) pattern of the synthesized UiO-66-NH. Characteristic diffraction peaks were observed at 7.34° and 8.48°, which are consistent with the diffraction peaks of UiO-66-NH. The sharp and narrow nature of the peaks indicates a high degree of crystallinity. Furthermore, the surface area and pore diameter of the synthesized UiO-66-NH were evaluated using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, respectively (data not shown). The surface area was calculated to be 984.218 m/g based on the BET equation, while BJH analysis suggested an average pore diameter of 2.968 nm. The obtained surface area and pore diameter were in good agreement with previously reported data for UiO-66-NH. The SEM, XRD, BET, and BJH results indicate the successful synthesis of UiO-66-NH with uniform particle morphology, high crystallinity, large surface area, and a nanoporous structure.
The adsorption of Pb onto UiO-66-NH was characterized using SEM, EDS, and SWASV. The SEM image revealed that there was no change in the octahedral-like morphology of UiO-66-NH after Pb adsorption (Fig. 5c), suggesting that the physical structure of the MOF remained intact during the adsorption process. In addition, EDS analysis of the Pb-adsorbed UiO-66-NH (MOF-Pb; Fig. 5d) exhibited peaks that were characteristic of O, Zr, Pb, C, and N. The weight% of Pb was measured at 6.81%, indicating successful Pbadsorption. SWASV was performed in acetate buffer (pH 4.5) to further confirm Pb adsorption. No oxidative peaks were observed for the pure MOF; in contrast, the MOF-Pb exhibited a well-defined Pb oxidation peak at a potential of -0.59 V (Fig. 5e). The results of the EDS and SWASV analyses indicate that Pb was successfully adsorbed onto UiO-66-NH.
The functionalization of CEA-specific aptamers onto UiO-66-NH was subsequently investigated using FT-IR, DLS, and zeta potential measurement. Figure 5f presents the FT-IR spectra of the synthesized MOF, MOF-Pb, and MOF-Pb-Apt. In the pristine MOF, characteristic peaks were observed at 1101 cm (C-N stretching of the -NH groups on the aromatic ring), 1656 cm (C = O stretching of the carboxyl groups in the linker), 3465 cm (N-H stretching of the -NH groups), and 768 cm (Zr-O vibrations). The weakening of C = O and N-H stretching at 1656 and 3465 cm was observed after Pb adsorption due to interaction between the metal ions and the amine and carboxyl groups of UiO-66-NH. In addition, the FT-IR spectrum of MOF-Pb-Apt exhibited a broad absorption peak at 1059 cm that corresponds to P-O stretching vibrations from the phosphate groups in the aptamer, indicating successful aptamer immobilization on the MOF-Pb composite. DLS analysis (Fig. 5g) revealed the hydrodynamic diameters of the MOF, MOF-Pb, and MOF-Pb-Apt to be 217.5 ± 10.8 nm, 254.4 ± 15.1 nm, and 264.9 ± 9.0 nm, respectively. The increases in particle size following Pb adsorption and subsequent aptamer functionalization once again confirm the successful modification of the MOF surface. Zeta potential measurements of MOF, MOF-Pb, and MOF-Pb-Apt are presented in Fig. 5h. The pristine MOF exhibited a positive zeta potential of + 51.3 ± 0.7 mV, which was attributed to the protonation of -NH/-NH- groups. Upon the adsorption of Pb, the zeta potential became negative (-23.1 ± 0.6 mV), indicating that the MOF-Pb had been successfully prepared. The negative zeta potential of MOF-Pb-Apt further increased to -38.5 ± 2.0 mV due to the negatively charged phosphate backbone of the aptamer. These results provide further confirmation of the successful adsorption of Pb onto UiO-66-NH as well as subsequent aptamer functionalization.
Key fabrication parameters that can influence the optimal performance of the sensor for CEA detection include hydroxyl group activation, APTES concentration, nanoMIPs concentration, and rebinding time; these parameters were systematically optimized to determine the ideal steps for the preparation of nanoMIPs/APTES/OH/MWCNTs/SPCE. Electrochemical measurements were conducted using a 2.5 mM [Fe(CN)] redox probe.
As hydroxyl group activation on the electrode surface was accomplished by using CV in NaOH solution, the number of CV scan cycles was optimized to maximize hydroxyl group generation. The number of scan cycles was varied across 0, 10, 30, and 50 cycles as demonstrated in Fig. 6a. There was a decreasing trend in current response as the number of CV scan cycles increased, highlighting the successful formation of hydroxyl groups on the electrode surface. However, the current response plateaued after 30 cycles, with no further significant decreases observed at 50 scan cycles. Consequently, 30 CV scan cycles in NaOH solution were determined to be the optimum for hydroxyl group activation.
The effect of APTES concentration on electrode modification was investigated using CV (Fig. 6b). Increasing current responses were observed as the APTES concentration increased to 3% v/v. However, further increases in APTES concentration to 10% v/v did not elicit any further significant enhancements in current response. The relatively stable current response beyond an APTES concentration of 3% v/v may be attributed to the limited surface area available for APTES functionalization on the electrode. Consequently, an APTES concentration of 3% v/v was selected as the optimal concentration for the preparation of APTES/OH/MWCNTs/SPCE.
The effect of nanoMIPs concentration on sensor performance was investigated by varying the concentration of nanoMIPs: 0.4, 0.6, 0.8, and 1.0 mg/mL of nanoMIPs were immobilized onto the APTES/OH/MWCNTs/SPCE. Each condition was tested with CEA concentrations of 0 ng/mL and 5 ng/mL; the corresponding SWV responses are presented in Fig. 6c. The results for the blank samples (CEA at 0 ng/mL) exhibited a decrease in current response with increasing nanoMIPs concentration due to the greater coverage of nanoMIPs on the electrode. These differences were normalized by evaluating the sensor performance in terms of the current change (ΔI) observed between the blank and CEA (5 ng/mL) samples (Fig. 6d). The highest ΔI was observed at a nanoMIPs concentration of 0.6 mg/mL, indicating that optimal sensitivity had been achieved. Higher concentrations of nanoMIPs did not promote CEA binding efficiency as indicated by the decrease in ΔI. Indeed, excessive nanoMIPs concentrations may result in loosely bound particles on the electrode surface, which could detach during the rebinding process, leading to a reduced or even negative ΔI. Therefore, 0.6 mg/mL nanoMIPs was chosen as the optimal concentration for sensor fabrication.
Finally, the rebinding time was varied (15, 30, 45, 60, and 90 min) to investigate its effect on sensor performance. Figure 6e shows that the SWV current responses decreased with increasing rebinding time, reflecting enhanced CEA binding to the nanoMIPs. However, saturation was observed at 60 min: extending the rebinding time to 90 min did not result in further significant improvements. Thus, a rebinding time of 60 min was chosen as the optimal condition for CEA detection.
For the preparation of the MOF-Pb-Apt signal probe, two critical parameters including lead ion concentration and aptamer concentration were optimized to enhance the sensor's performance.
Since the amount of Pb loaded affects the sensitivity of the signal response, the concentration of Pb was optimized to achieve the best performance of the proposed sensor. UiO-66-NH was loaded with Pb at concentrations of 1, 10, 30, and 50 mM. After removing the excess Pb, the synthesized MOF-Pb prepared at different Pb concentrations was characterized using SWASV in acetate buffer (pH 4.5). Figure 6f compares the oxidative current responses of Pb from MOF-Pb prepared at different loading concentrations. As expected, the current response increased with Pb concentration from 1 to 30 mM. However, a further increase to 50 mM did not result in any significant change compared with 30 mM. This plateau in current response can be attributed to the limited number of active sites on UiO-66-NH. Therefore, 30 mM Pb was selected as the optimal concentration for signal probe preparation.
As the aptamer is the key element for the specific binding of the signal probe toward the target CEA, its concentration was optimized to improve sensor performance. The synthesized MOF-Pb was functionalized with the CEA-specific aptamer at concentrations of 0, 100, 300, 500, and 800 nM, and the corresponding zeta potential values were compared (Fig. 6g). A more negative zeta potential was observed as the aptamer concentration increased from 100 to 500 nM, indicating a higher amount of aptamer immobilized onto UiO-66-NH. However, no significant change in zeta potential was observed when the concentration was further increased to 800 nM, which can be attributed to the limited surface area of UiO-66-NH. Therefore, 500 nM was selected as the optimal aptamer concentration for immobilization.
Following the successful preparation of the nanoMIPs/APTES/OH/MWCNTs/SPCE and the signal probe (MOF-Pb-Apt), both components were subsequently used in a nanoMIPs-aptamer sandwich assay for CEA detection. The platform was tested using CEA concentrations of 0 and 50 ng/mL; the results were compared to a control system using a nanoNIPs-aptamer sandwich assay. SWASV was performed to measure the oxidative current response of Pb, which corresponds to the amount of CEA bound to the electrode surface. Figure 7a presents the results obtained from the nanoMIPs-based platform. Oxidative current responses of Pb were detected after exposure to both 0 and 50 ng/mL CEA. The response at 0 ng/mL CEA was considered to be the background response; this represents the residual template CEA molecules that remained following the polymerization process. There was a clear increase in current response in the sample with 50 ng/mL CEA, suggesting that CEA had been successfully bound to the imprinted sites, facilitating the formation of the sandwich complex. In contrast, the control experiment using the nanoNIPs-based platform (Fig. 7b) exhibited minimal current responses at both concentrations. Although a slight increase in the current response of the 50 ng/mL CEA sample was observed, this was likely due to non-specific adsorption. These findings demonstrate the efficiency of the nanoMIPs-aptamer sandwich assay for CEA detection.
The performance of the developed sensor for CEA detection was systematically evaluated under optimized conditions. A range of CEA concentrations from 1 to 1000 ng/mL was tested; each was followed by the addition of MOF-Pb-Apt to form the sandwich complex. SWASV measurements were performed in acetate buffer (pH 4.5). The resulting voltammograms corresponding to each CEA concentration are shown in Fig. 7c and d for the nanoMIPs-based and nanoNIPs-based platforms, respectively. The corresponding calibration curves between the current responses and the CEA concentrations (log-scale) are presented in Fig. 7e. The oxidative current response of the Pb ions obtained from the nanoMIPs-based platform increased significantly with increasing CEA concentration, suggesting that the signal originated from the specific binding of CEA to the nanoMIPs-based recognition sites. The developed sensor exhibited a linear detection range from 1 to 1000 ng/mL, represented by a linear trend that can be described by the regression equation I (µA) = 8.8001 × log[CEA] + 15.134 (R = 0.9894). In contrast, only a slight increase in the current response of Pb ions was observed for the nanoNIPs-based platform. The corresponding linear regression equation was found to be I (µA) = 0.5562 × log[CEA] + 2.499 with a correlation coefficient of R = 0.6598. Statistical analysis revealed that the significance of the correlation had a p-value of 0.00795. The sensitivity of the nanoMIPs-based platform was found to be approximately 15.8 times higher than that of the nanoNIPs-based platform, clearly demonstrating the efficient and selective binding of the nanoMIPs sensor toward CEA. The limit of detection (LOD) was determined to be 1.4 ng/mL, calculated using the formula 3σ/slope, where σ represents the standard deviation of the blank measurements. The higher LOD compared to the lowest experimentally tested concentration within the linear range can be attributed to baseline variation and the moderate slope. However, this LOD is lower than the cut-off value of 5 ng/mL for clinical CEA diagnostics, highlighting the potential applicability of the proposed sensor for CEA detection.
The linear detection range and LOD obtained in this study were comparable to or better than those reported for previously published electrochemical MIP-based sensors for CEA detection (Table 1). Although the LOD reported in this study is not the lowest among previous works, the obtained linear detection range covers the clinically relevant concentration range (5 ng/mL to 1000 ng/mL), typically observed in cancer patients. This wide detection range eliminates the need for tedious sample dilution steps. In contrast, most previously reported electrochemical MIP-based sensors for CEA detection primarily relied on label-free strategies. These studies focused on enhancing sensor sensitivity by modifying electrode surfaces with various nanomaterials and incorporating MIPs to provide specific recognition sites for CEA. However, their detection principle was largely based on the inhibition of electron transfer of redox compounds, such as Ru (III), iodine, and the commonly used ([Fe(CN)]) probe. Apart from using redox probes in the electrolyte, cobalt-based metal-organic frameworks (Co MOFs) have also been reported as modifier on SPCEs to serve as detection signals. Although electrochemical label-free detection is convenient and widely used, nonspecific interferences in complex biological samples have been reported, leading to reduced analytical performance. In contrast, the nanoMIPs-aptamer sandwich assay presented in this work enables the incorporation of a signal probe into the detection system. The use of a signal probe not only enhances the sensor's sensitivity but also provide more selective recognition of CEA. In addition, most previously reported electrochemical MIP-based sensors for CEA detection employed electropolymerization to produce thin-film MIP on the electrode surface. Although this technique is suitable for protein imprinting and can be applied to a variety of electroactive monomers, it requires polymerization on each individual electrode. This not only results in poor reproducibility but also makes the process time-consuming and challenging to scale up for large-scale production. In addition to thin film MIPs, core-shell molecular imprinting has also been proposed to improve the sensitivity of CEA detection. Although this technique addresses the challenge of mass production, the signal measurement still relies on the inhibition of electron transfer of redox compound, [Fe(CN)]. In contrast, the present work employs nanoMIPs synthesized via solid-phase synthesis, which addresses the challenges associated with mass production. The CEA-specific nanoMIPs can be synthesized on a large scale for sensor fabrication, thereby overcoming the problem of poor reproducibility. Moreover, using nanoMIPs instead of thin-film MIP avoids the time-consuming process of large-scale sensor fabrication, as nanoMIPs can be immobilized on multiple electrodes simultaneously. In addition, since nanoMIPs are in nanoparticle form, unlike rigid MIP films on electrode surfaces, they can be applied to a variety of transducers, enabling broader applications. Furthermore, the sandwich format allows for the integration of signal probe, making this platform adaptable for multiplex biomarker detection, which remains a significant challenge for conventional MIP thin-film sensors.
The selectivity of the proposed sensor was investigated by comparing its response toward CEA as well as other potential interferences. The nanoMIPs-aptamer sandwich assay was performed on a blank sample, 5 and 50 ng/mL CEA samples, human serum albumin (HSA, 0.1 mg/mL), immunoglobulin G (IgG, 0.1 mg/mL), and cancer antigen 15 - 3 (CA 15 - 3, 30 U/mL). Their respective oxidative current responses are presented in Fig. 8a. There was a noticeable increase in the oxidative current response of the CEA samples compared to the blank; in contrast, no significant changes were observed in the presence of any other interfering substances. These findings highlight the high selectivity of the proposed sensor toward CEA.
Reproducibility is a critical component of sensor development. The reproducibility of the proposed nanoMIPs-aptamer sandwich assay was evaluated using 12 independently fabricated sensors. Each sensor was tested on a 50 ng/mL CEA sample. Figure 8b shows that all sensors exhibited highly consistent responses with a low relative standard deviation (RSD) of 2.97%. This finding highlights the high reliability and consistent response of sensors fabricated using the methods proposed in this work.
The nanoMIPs-aptamer sandwich assay was applied to the detection of CEA in human serum samples. Commercial human serum was spiked with CEA at concentrations of 5, 10, and 25 ng/mL. Although the proposed sensor demonstrated satisfactory selectivity toward CEA, it is important to note that the concentration of human serum albumin (HSA) in real human serum is considerably high (35-50 mg/mL). Such high levels of HSA may interfere with the biosensor response through nonspecific binding or surface blocking. To address this issue, a pretreatment step using a molecular weight cut-off (MWCO) filter was applied to reduce the HSA concentration in the serum samples. After pretreatment to minimize the effects of HSA, the samples were analyzed using the prepared sensor to evaluate its performance in clinical applications. The RSD values of the responses ranged from 3.68 to 7.63%, and the average recoveries obtained were between 98.12 and 103.24%. Statistical t-tests revealed no significant differences between the spiked concentrations and the measured values at a 95% confidence level (p-value ≥ 0.05; Table 2). These results emphasize the potential application of the developed sensor to the accurate and precise detection of CEA in human serum samples.